The search for new classes of compounds for the therapy and prophylaxis of proliferative diseases, cancer and metabolic deregulation is one of the most important tasks for pharmaceutical research. These diseases affect a large portion of the population, leading to suffering and often being the cause for the death of the individuals stricken therewith.
Signal transduction is the process of relaying extracellular messages, e.g. chemical messages in the form of growth factors, hormones and neurotransmitters, via receptors, e.g. cell-surface receptors, to the interior of the cell. At the heart of this biological communication are the protein-tyrosine kinases. These enzymes, found, for example, as either transmembrane growth factor receptors or as nuclear or cytosolic non-receptor proteins, catalyze the phosphorylation of specific tyrosine residues. This class of enzymes includes, but is not limited to, the PDGF receptor, the FGF receptor, the HGF receptor, members of the EGF receptor family such as the EGF receptor, the HGF receptor, members of the EGF receptor family such as the EGF receptor, erb-B2, erb-B3 and erb-B4, the src kinase family, Fak kinase and the Jak kinase family. The tyrosine-phosphorylated proteins are involved in a range of metabolic processes, from proliferation and growth to differentiation. Protein-tyrosine phosphorylation is known to be involved in modulating the activity of some target enzymes as well as in generating specific complex networks involved in signal transduction via various proteins containing a specific amino acid sequence called a Src Homology region or SH2 domain (for review see Proc. Natl. Acad. Sci. USA 90, 5891 (1990)). A malfunction in this protein-tyrosine phosphorylation through tyrosine kinase overexpression or deregulation is manifested by various oncogenic and (hyper-)proliferative disorders such as cancer, inflammation, autoimmune disease, hyperroliferative skin disorders, such as psoriasis, and allergy/asthma.
SH2- and/or SH3-comprising proteins that play a role in cellular signaling and transformation include, but are not limited to, the following: Src, Lck, Eps, ras GTPase-activating protein (GAP), phospholipase C, phosphoinositol-3 (Pl-3)kinase, Fyn, Lyk, Fgr, Fes, ZAP-70, Sem-5, p85, SHPTP1, SHPTP2, corkscrew, Syk, Lyn, Yes, Hck, Dsrc, Tec, Atk/Bpk, Itk/Tsk, Arg, Csk, tensin, Vav, Emt, Grb2, BCR-Abl, Shc, Nck, Crk, CrkL, Syp, Blk, 113TF, 91TF, Tyk2, esecially Src, phospholipase c, phoshoinositol-3 (pl-3) kinase, Grb2, BCR-Abl, Shc, Nck, Crk and CrkL.
A direct link has been established between activated receptor kinases and Ras with the finding that the mammalian Grb2 protein, a 26 kilodalton protein comprising a single SH2 and two SH3 domains bind to proline-rich sequences present in the Sos exchange factor.
The significance of ras-regulatory proteins in human tumors is also highlighted by the critical role of GRB2 in BCR-Abl mediated oncogenesis (J. Exp. Med., 179(1), 167-175 (1994)).
Recently, DNA sequences within the chromosomal locus 17q22-qter, which harbors the GRB2 gene, were shown by comparative genomic hybridization to exhibit a high frequency of amplification both human breast cancer cell lines and tumors (Proc. Natl. Acad. Sci. USA 91, 2156-2160 (1994)).
In a study of GRB2 gene expression in human breast cancer cell lines, Northern Blot analysis also revealed that 7/19 breast cancer cell lines exhibited more than 2 fold overexpression of GRB2 MRNA relative to normal breast epithelial cells. In MCF-7, MDA-MB-361, and -453 cells, the overexpression of GRB2 MRNA was accompanied by a 10-20 fold increase in the amount of GRB2 protein (Oncogene 9, 2723 (1994)).
SH2 domains represent recognition motifs for specific tyrosine-phosphorylated peptide sequences. Short, conserved motifs, primarily 3 to 6 amino acid on the carboxy-terminal side of phosphotyrosine residue, carry the sequence-specific information for SH2-recognition. This concept has been supported by the mapping of separate sites for binding of SH2 domains from different signaling molecules on various receptors [see, e.g., Cell 69, 413 (1992); Proc. Natl. Acad. Sci. USA 89, 678 (1992); Mol. Cell. Bio. 12, 991 (1992); EMBO J. 11, 1365 (1992); EMBO J. 11, 559 (1992); EMBO J. 11, 3911 (1992): Cell 73, 321 (1993)]. Degenerate peptide libraries have also been used to predict the specifity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-.gamma., p85 subunit of Pl-3 kinase, and HCP (amino terminal SH2) [see Cell 72, 767 (1993); Mol. Cell. Biol. 14, 2777 (1994)]). High-resolution crystallographic analysis and nuclear magnetic resonance of the SH2 domains of Src, Lck, PLC-.gamma. C-terminal, p85 N-terminal, Abl, Syp C-terminal have also revealed that the region on the carboxyl side of the phosphotyrosine carries the sequence-specific information for SH2 recognition. Each of these SH2 containing proteins controls a cellular pathway involved in the biological response to a growth factor. Activation of a particular pathway can thus be inhibited by designing a small molecule that specifically disrupts a phosphoprotein/SH2 domain interaction.
Src homology 2 (SH2) domains are protein motifs which recognize and bind with high affinity to phosphotyrosyl (pTyr, 1)-containing proteins. These modules serve critical roles in protein-tyrosine kinase (PTK) pathways by protein-protein oligomerization in response to specific signalling events..sup.1 The growth factor receptor-bound 2 (Grb2) SH2 domain is a key link in mitogenic Ras pathways, functioning as a bridging element between cell surface growth factor receptors and the Ras protein. Grb-2 mediated activation of Ras pathway is highly relevant to a number of diseases, including breast cancer, where members of the epidermal growth factor receptor (EGFR) PTK family such as erbB-2 (HER-2/neu) are frequently over-expressed..sup.2 Progress in the development of novel high affinity Grb2 SH2 domain ligands.sup.3-5 is typified by 11,.sup.6 whose design is based on binding of a larger pTyr-containing peptide to the Grb2 SH2 domain in .beta.-bend fashion..sup.7 While these inhibitors take advantage of high affinity interactions with SH2 domains outside the pTyr-binding pocket, finding suitable phosphatase-resistant replacements for the pTyr residue itself has continued to be a challenge..sup.8 In the area of non phosphorus-containing phosphate alternatives, analogues (9) of 4,5-dideoxyshikimate-3-phosphate (8) first demonstrated the utility of the malonate group as a phosphate mimetic..sup.9. Based on this work, the dicarboxylic-containing O-malonyl-L-tyrosine (OMT, 4).sup.10 was prepared on a non-phosphorus-containing pTyr mimetic. Although OMT residues are moderately potent against a variety of SH2 domains,.sup.11 they share with parent pTyr the disadvantage of two negative charges. Efforts are therefore underway to identify monocarboxylic-based pTyr mimetics for use in Grb2 SH2 domain antagonists. ##STR2##
Signal transduction is critical to normal cellular homeostasis, with abberations in some signalling pathways having potentially adverse effects, including the promotion of cancers and immune disorders. For many growth factor and cytokine pathways, binding of extracellular ligands results in intracellular signalling through the phosphorylation of tyrosyl residues by protein-tyrosine kineses (PTKs). This can result in subsequent signal transduction through protein-protein binding mediated by Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains, which recognize with high affinity, specific pTyr-containing sequences..sup.12,13 The phosphotyrosyl pharmacophore (pTyr, 1) serves a central role by providing key recognition features necessary for assembly of these multi-component signalling complexes..sup.14 It has long been realized that inhibitors of abberant pTyr-dependent signalling could potentially afford new therapeutic approaches..sup.15 Since pTyr-dependent signalling is comprised of three distinct interacting components; (1) generation of pTyr residues by PTKs, (2) formation of protein complexes through SH2 and PTB domain-mediated pTyr-dependent processes and (3) destruction of pTyr residues by protein-tyrosine phosphatases (PTPs), inhibitors could potentially be directed at any of these three points..sup.16 While initial work was focused on development of PTK antagonists,.sup.17-19 PTPs have emerged as more recent targets..sup.20 Both of these latter approaches rely on enzyme inhibition to achieve their effects. Alternatively, antagonizing pTyr-dependent signalling could result through the direct prevention of SH2 and PTB domain binding processes..sup.21,22
Central to the binding of SH2 domains with pTyr-containing ligands is the interaction of the doubly ionized pTyr phosphate with two invariant arginine residues in a well formed pocket..sup.23-33 These arginine-phosphate interactions are particularly critical to the overall binding, such that high affinity binding is usually lost by removal of the phosphate group. Additional, secondary binding interactions are provided by amino acids 2-3 residues C-proximal to the pTyr residue, which introduce differential affinity toward SH2 domain sub-families..sup.23 Design of SH2 domain inhibitors therefore concerns itself with three thematic areas: (1) interactions within the pTyr 3 binding pocket.sup.16 (2) interactions with recognition areas outside the pTyr pocket and (3) bridging elements between structural features (1) and (2). Examples of this type of approach have recently been reported. .sup.35,36
Although the pTyr pharmacophore plays a dominant role in SH2 domain.ligand interactions, pTyr residues are not suitable components of inhibitors intended for in vivo application, due to the enzymatic lability of the phosphate ester bond and to the poor cellular penetration of doubly ionized phosphate species. Design of pTyr mimetics for use in SH2 domain antagonists has therefore focused on agents which are stable to phosphatases and which offer the potential for cell membrane penetration. Phosphonate-based pTyr mimetics, such as phosphonomethyl phenylalanine (Pmp, 2).sup.37 and difluorophosphonomethyl phenylalanine (F.sub.2 Pmp, 3).sup.38-40 replace the tyrosyl phosphate ester bond with a methylene unit. These analogues are stable to phosphatases, retain good SH2 domain binding affinity,.sup.16,41,42 and when administered into cells by microinjection.sup.43 or cell permeabilization techniques.sup.44 have been shown to exhibit effects consistent with SH2 domain inhibition. Similarly, efficacy has been shown during short term incubation of cells with pTyr-peptides containing special membrane transport sequences which deliver charged species into cells..sup.45,46 However, while useful for pharmacological studies, such delivery techniques do not hold promise for in vivo studies. A more traditional approach using prodrug derivatization of the phosphonate moiety has met with limited success when applied to F.sub.2 Pmp-containing peptides..sup.47 These considerations have lead to the examination of non-phosphorus containing pTyr mimetics which may offer alternatives to cell delivery..sup.16 48,49 Among such analogues are O-malonyltyrosine (OMT, 4).sup.50 and fluoro-o-malonyltyrosine (FOMT, 5),.sup.51 which utilize malonate or fluoromalonate groups to mimetic the tyrosyl phosphate. The present study was undertaken to examine both known pTyr mimetics such as F.sub.2 Pmp and Pmp as well as new pTyr mimetics, in the contex of Grb2 (growth factor receptor bound 2).sup.52 SH2 domain binding systems. The relevance of Grb2 to several cancers, including breast cancer, highlights the need for greater diversity in ligands which disrupt these signalling processes. ##STR3##
It is a goal of the present invention to present small organic molecules that, due to their ability to mimetic the structure of the phosphotyrosine peptide binding site, have the ability to disrupt the interaction between SH2 domains of (e.g. regulatory) proteins, for example that of Grb2, and proteins with phosphorylated moieties, especially phosphorylated tyrosine moieties, for example phosphorylated protein tyrosine kinase receptors. The effect is to inhibit the associated of SH2 containing (e.g. regulatory) proteins with a protein tyrosine kinase in order to inhibit downstream signalling through one or more specifically targeted effector proteins.